The goal of this project is to test if rhesus cytomegalovirus-based SIV vaccines (RhCMV/SIV) can be improved via IL-10 pathway modulation to obtain significantly over 50% protective efficacy. We found that second-generation RhCMVdIL10/SIV vaccines lacking the viral IL-10 gene protect non-human primate infants, while first-generation (IL-10 intact) RhCMV/SIV vaccines do not. First-generation vaccines have been proven effective only in wild-type RhCMV-seropositive macaques having neutralizing antibodies to viral IL-10. The significance of this work is that if successful it will provide (i) new candidate HIV vaccines with greater efficacy in some or all segments of the population, (ii) a coherent, mechanistic explanation for previously obscure patterns of RhCMV/SIV vaccine protectiveness, (iii) new pharmacologic tools for control over IL-10 signaling, and (iv) immunologic insight into consequences of cellular and viral IL-10 pathway modulation. Our preliminary data show that rhesus macaques infected by wild-type rhesus cytomegalovirus (wtRhCMV) mount immune responses to viral interleukin-10 (vIL-10), which in most cases leads to generation of neutralizing antibodies. RhCMV/SIV vaccine stringently protects ~50% of such wtRhCMV+ but not wtRhCMV- seronegative monkeys against SIV challenge. A second-generation RhCMV/SIV vaccine lacking the viral IL-10 gene, however, can protect seronegative macaques. We hypothesize that RhCMV/SIV vaccination in the context of inhibited host IL-10 signaling will achieve superior efficacy (>50%) in infant and adult macaques. Our specific aims are: Aim 1. Define the transcriptomic and immunologic signatures of increased or decreased IL-10 signaling using previously collected samples. Here we determine the true transcriptomic signature of host IL-10 signaling using samples from animals receiving anti-IL-10 antibody or with forced IL-10 expression. We then evaluate host responses to RhCMV/SIV vaccination in the presence of varying levels of viral IL-10 neutralization to determine how such neutralization affects (i) host IL-10 signaling, (ii) likelihood of generating a transcriptomic signature associated with protection, and (iii) vaccine efficacy. Aim 2. Test if cellular IL-10 inhibition augments vaccine efficacy in infant (RhCMV-negative) macaques. Since viral IL-10 deletion presumably interferes with the host IL-10 response, we reason that further interference (via administration of neutralizing anti-IL-10 antibody) will further augment vaccine efficacy. In this aim we therefore administer RhCMVdIL10/SIVgag alone or in the presence of neutralizing anti-cIL-10 antibody. Aim 3. Test if cellular and/or viral IL-10 inhibition augment vaccine efficacy in adult (RhCMV-positive) macaques. Here we test if complete inhibition of viral and/or cellular IL-10 can substantially increase efficacy. Together these studies will provide clear understanding of the role of IL-10 signaling in RhCMV/SIV vaccine efficacy and may point the way to new vaccine regimens that are 85-100% effective.